Transgenic pathogen-resistant organism

ABSTRACT

Transgenic pathogen-resistant organism whose genome contains at least two different genes under the control of active promoters with pathogen-inhibiting action. This organism is distinguished by a synergistic pathogen-inhibiting action. This action is evident particularly when the genes code for the gene products chitinase (ChiS, ChiG), glucanase (GluG), protein synthesis inhibitor (PSI) and antifungal protein (AFP).

[0001] Transgenic pathogen-resistant organism

[0002] The invention relates to a pathogen-resistant organism and to aprocess for generating it.

[0003] It is known in the state of the art that infestation of a plantby pathogens are caused a series of different reactions. These include,for example, changes in the cell wall structure, the synthesis ofphytoalexins which have antimicrobial activity, the accumulation ofso-called PR proteins (pathogenesis-related), protease inhibitors andenzymes with hydrolytic functions (Hahlbrock and Grisebach in Ann. Rev.Plant. Physiol., 30 (1979), 105-130).

[0004] Many pathogens (fungi and insects) have chitin as a constituentof their cell wall. By contrast, plants possess no chitin. It has nowbeen demonstrated in some cases that there is enhanced production ofchitinases in plants after infestation by pathogens. Chitinases areamong the enzymes with hydrolytic functions and they catalyze chitinbreakdown. It has now been possible to show that plants acquire anincreased resistance to pathogens by the production of chitinases.

[0005] It is furthermore known to use a gene from barley plants whosegene product codes for an inhibitor of fungal protein synthesis. Theincorporation of a corresponding inhibitor gene in transgenic plants ledto improved resistance to fungi.

[0006] Finally, it has also been disclosed that the use of a polypeptidefrom Aspergillus giganteus is able to protect, by virtue of itsantifungal activity, plants from infestation by fungi.

[0007] However, given this state of the art there is a need to providefurther transgenic pathogen-resistant organisms. Moreover, the organismswhich are particularly desired are those whose resistance is increasedoverall by comparison with the known organisms or is extended withrespect to the number of possible pathogens.

[0008] This problem is solved by a transgenic pathogen-resistantorganism having the features of claim 1.

[0009] The invention is based on the surprising finding that theincorporation of at least two different genes with pathogen-inhibitingaction into the genome of an organism assists the latter to resistpathogens to an extent going far beyond an additive effect of each ofthe genes on its own.

[0010] The dependent claims indicate further embodiments of theinvention.

[0011] The genes can code for gene products which reduce the vitality offungi. In particular, the genes can be of fungal, bacterial and plant,animal or viral origin. In particular, the gene products have propertieswhich promote resistance to fungi. The gene products are chitinase(ChiS, ChiG), glucanase (GluG), protein synthesis inhibitor (PST) andantifungal protein (AFP).

[0012] The transgenic pathogen-resistant organism can be a plant, andtobacco, potato, strawberry, corn, rape or tomato plants are preferred.

[0013] The invention also relates to DNA-transfer vectors with insertedDNA sequences as are indicated in detail in this description.

[0014] The invention Furthermore relates to a process for the generationof pathogen-resistant organisms as are described herein, wherein atleast 1 gene with pathogen-inhibiting action is transferred into thegenome of an organism, and the pathogen-resistant organism is obtained(a) by crossing the organism with another, optionally transgenic,organism which contains at least one other gene with pathogen-inhibitingaction, and subsequently selecting, and/or (b) by transformation of thisother gene with pathogen-inhibiting action into the organism. Theprocess can be used with DNA-transfer vectors with inserted DNAsequences corresponding to a gene with pathogen-inhibiting action asdescribed herein.

[0015] Finally, the invention relates to a process for the generation ofpathogen-resistant organisms, wherein vectors which comprise more thanone gene with pathogen-inhibiting action are used for the transformationinto the genome of an organism.

[0016] The invention also relates to a process for ensuring theresistance of organisms to pathogens, characterized in that the organismused is a transgenic pathogen-resistant organism according to any orclaims 1 to 7 or an organism whose genome contains at least one genecomplying with the definitions used herein (see claims 1 to 7, and atleast one substance which is not expressed by the organism butcorresponds to any other one of the gene products complying with thedefinitions given in this application (claims 1 to 7) is applied to theorganism.

[0017] It was possible to achieve the synergistic effects veryparticularly with transgenic pathogen-resistant organisms to which thegene sequences which coded for proteins of the attached sequencelistings A to E, or corresponded to the latter, were transferred ortransfected.

[0018] ChiS

[0019] A DNA fragment which is 1.8 Kb in size, that codes for achitinase called ChiS was isolated from the soil bacterium Serratiamarcescens. In vitro investigations with purified ChiS protein showedthat it is able effectively to inhibit the growth of fungi, even in lowconcentrations. The reason for the inhibition is that the ChiS proteinhas a chitinase activity which is able to damage the tips of the fungalhyphae. In this way the fungus is unable to grow further and isinhibited.

[0020] PSI

[0021] The PSI gene originates from barley and codes for a protein whichinhibits protein synthesis by fungi. In vitro tests show that even lowconcentrations of PSI are sufficient to inhibit various fungi such as,for example, Rhizoctonia solani.

[0022] AFP

[0023] It is possible for a polypeptide which has antifungal activity tobe isolated from the fermentation broth of Aspergillus giganteus and tobe sequenced. This polypeptide is suitable as antifungal agent, forexample as spraying agent and as preservative for industrial productsand human and animal foods. It can furthermore be combined with othersubstances which have pesticidal activity, fertilizers or growthregulators. Inhibitory activities against fungi were detectable interalia against various Aspergillus, Fusaria, Phytophthora and Trichophytonspecies.

[0024] ChiG and GluG

[0025] Two genes which code, respectively, for a chitinase (ChiG) andglucanase (GluG) can be isolated from certain types of barley. PurifiedChiG protein or GluG protein inhibits various phytopathogenic fungi invitro (inter alia Rhizoctonia solani) (see R. Leah et al., Journal ofBiological Chemistry, Vol. 266, No. 3 (1991), pages 1564-1573).

[0026] The inventors have now found, completely surprisingly, that an atleast binary combination of expression of PSI, AFP, ChiS, ChiG or GluGleads to synergistic effects in respect of the acquired resistance tofungi in transgenic plants. In particular, the effects of the individualsubstances in the combination are markedly exceeded. These includeresistance to the fungus Rhizoctonia solani, Sclerotinia infestation,Botrytis infestation, etc.

[0027] Combinations according to the invention are (DNA and/orpolypeptides):

[0028] binary combinations

[0029] ChiS, GluG; ChiS, PSI; ChiS, ChiG; ChiS, AFP; GluG, PSI; GluG,ChiG; GluG, AFP; PSI, ChiG; PSI, AFP;

[0030] ternary combinations

[0031] ChiS, GluG, PSI; ChiS, GluG, ChiG; ChiS, GluG, AFP; GluG, PSI,ChiG; GluG, PSI, AFP; PSI, ChiG, AFP; ChiG, AFP, GluG

[0032] quaternary combinations

[0033] ChiS, GluG, PSI, AFP; ChiS, GluG, PSI, ChiG;

[0034] quinary combination

[0035] ChiS, GluG, PSI, AFP, ChiG

[0036] The invention furthermore relates to the combined use of theproteins with pathogen-inhibiting action, preferably ChiS, PSI, AFP,ChiG and GluG, against pathogens. Combined use also means in thiscontext that at least a first pathogen-inhibiting substance is expressedby the organism and at least a second substance which haspathogen-inhibiting action is applied to the organism from outside.

[0037] The agents according to the invention also include those whichcontain the abovementioned proteins in at least binary combination. Theagents according to the invention can contain other active substancesbesides the proteins. The other active substances can be pesticides,fertilizers and/or growth regulators, and the agents according to theinvention can be prepared in various formulations such as concentrates,emulsions, powders, formulations on carriers, mixtures with other activesubstances, etc. The ChiS/PSI and AFP/PSI combination is particularlypreferred. These proteins can be used particularly effectively toinhibit the growth of Rhizoctonia solani, especially in tobacco crops.

[0038] The invention also relates to the use in a process according tothe invention of a DNA sequence which codes at least for a polypeptideof sequences A to E, or to a pathogen-resistant organism, where itsgenome contains at least two different genes under the control of activepromoters with pathogen-inhibiting action, where the genes are in eachcase selected from the group of sequences A to E. The inventionfurthermore includes DNA sequences which hybridizes with a DNA sequencewhich codes for polypeptides of amino-acid sequences A to E; where theseDNA sequences can be of natural synthetic [sic] or semisynthetic originand can be related to the abovementioned DNA sequence by mutations,nucleotide substitutions, nucleotide deletions, nucleotide insertionsand inversions of nucleotide sequences, and codes for a polypeptide withpathogenic activity. The invention furthermore relates to a recombinantDNA molecule which contains at least one DNA sequence which accords withthe preceding statements, where this DNA molecule can be in the form ofa cloning or expression vector.

[0039] The invention relates to appropriate host organisms andintermediate hosts which are transformed with a recombinant DNA moleculewhich accords with the preceding statements. Preferred as intermediatehost in the generation of a pathogen-resistant transgenic organism arestrains of bakeria, in particular so-called Agrobakeria strains.

[0040] The invention furthermore relates to the transgenicpathogen-resistant organisms obtained by the process according to theinvention, in particular tobacco, potato, corn, pea, rape and tomatoplants.

[0041] The DNA sequences according to the invention are, as a rule,transferred together with a promoter. Promoter sequences are recognizedby the plant transcription apparatus and thus lead to constitutiveexpression of the gene associated with them in plants. The promoter can,however, also be pathogen-inducible and/or wound-inducible (WUN1) and/ortissue-specific and/or development-specific.

[0042] The genetic manipulation operations necessary for carrying outthe invention, especially for expression of the gene in plants, aregenerally known. See for example the publication by Maniatis et al. in“Molecular cloning: A laboratory manual”, Cold Spring Harbor (1982).

[0043] The invention is explained in detail in the following examples.

[0044] All the standard methods of molecular biology were carried out,unless otherwise indicated, as described by Maniatis et al. “Molecularcloning: a laboratory manual”, Cold Spring Harbor (1982).

[0045] The DNA coding for amino-acid sequences A to E was initiallycloned in a manner known per se and then transferred by conjugation intoA. Tumefaciens LBA 4404 (A. Hoekema et al., Nature 303, 179-180). Thistook place by the method described by Van Haute et al. in EMBO J. 2,411-418 (1983).

[0046] The transfer of DNA into that Agrobacterium was checked byisolating Agrobacterium DNA by the method described by Ebert et al. inProc. Natl. Acad. Sci. USA 84 5745-5749 (1987). Restriction cleavage ofthe DNA, transfer to Hybond-N membrane (Amersham) and hybridization witha radioactively labeled DNA probe provided information about successfulDNA transfer into the Agrobacterium.

[0047] The transformed Agrobacterium was then used to transform tobaco,rape, strawberry, tomato and potato plants.

[0048] The LBA4404 Agrobacteria required for the infection wereinitially cultivated in selective antibiotic medium (P. Zambrisky et al.in EMBO J., 1, 147-152 (1983)), sedimented by centrifugation and washedin YEB medium without antibiotics (YEB=0.5% meat extract; 0.2% yeastextract; 0.5% peptone; 0.5% sucrose; 2 mM MgSO₄). After renewedsedimentation and taking up in MgSO₄ it was possible to use the bacteriafor the infection.

[0049] The so-called leaf disk method was used for the infection.

[0050] Sterile leaves were used for the leaf disk infection. Leaf piecesabout 1 cm in size are dipped in the previously described Agrobacteriasuspension and subsequently transferred to 3MS medium (medium describedby T. Murashige and F. Skoog in Physiol. Plant., 15, 473-497 (1962);3MS=MS−3% sucrose). After incubation at 25° C. to 27° C. with 16 hoursof light for two days, the leaf pieces were transferred to MSC16 medium(according to T. Murashige (see above); MSC16=MS+0.5 μg/ml BAP+0.1 μg/mlNAA+100 μg/ml kanamycin sulfate+500 μg/ml Claforan). Shoots appearingafter 4-6 weeks were cut off and transplanted to MSC15 medium (accordingto Murashige (see above); MSC15=MS+2% sucrose, 500 μg/ml Claforan+100μg/ml kanamycin sulfate). Shoots with root formation were analyzedfurther.

[0051] Monocotyledonous plants (including corn), but some dicotyledonousplants too, were transformed by direct gene transfer into protoplasts.These protoplasts were subsequently regenerated to intact plants(Example: J. Potrykus in Biotechnology 8 (1990), 535).

[0052] The resulting transgenic plants were infected with the fungusRhizoctonia solani for testing purposes. For this purpose, fungalcultures were grown and thoroughly mixed in standard soil. This soil wasthen distributed in a dish and planted with the plants to be tested.

[0053] For the evaluation, each plant on a dish was assigned a valuefrom 0 to 3. It was possible to calculate from this for each plant linean index which resulted from the sum of the values. The classificationis as follows:

[0054] 0=no symptoms (healthy)

[0055] 1=slightly reduced size (compared with a non-infected control);no or very slight visible infestation

[0056] 2=severe reduction in growth; severe symptoms of infestation

[0057] 3=dead

[0058] The rating is carried out in each case 14 days after the start ofthe series of tests.

EXAMPLE 1

[0059] Fungus inhibition test with combined proteins

[0060] The intention initially was to show that the proteins used herehave synergistic effects in their combination. Fungal growth tests invitro were carried out for this purpose.

[0061] These entailed a defined amount of Rhizoctonia solani fungalmycelium being mixed with 100 μl of potato dextrose solution andincubated in microtiter plates at 25° C. In this test there is a linearcorrelation between the growth of the fungus and the increase in theoptical density at 405 nanometers. The inhibitory effect of proteins canbe detected from a smaller increase in the optical density.

[0062] 2-3 mycelium balls were taken from a liquid culture of R. Solani,mixed with 100 μl of KGB medium in an Eppendorf vessel and carefullyhomogenized with a glass mortar. This suspension was then mixed with 10ml of KGB medium and passed through a sterile 100 μm screen. The opticaldensity of this mycelium fragment suspension (100 μl aliquot) wasadjusted to a value of 0.06-0.07 at 405 nanometers by adding medium. 100μl samples were placed on a microtiter plate and mixed with the proteinsto be tested. 7 parallels were measured per mixture. Mixtures which weremixed with the corresponding amounts of buffer served as controls. Theplates were incubated in the dark at 25° C. for 48 hours, and theoptical density of the cultures was measured at regular intervals.

[0063] Calculation of whether two proteins act together in an additivesynergistic or antagonistic manner in the inhibition of fungal growth ispossible from the measured data with the aid of the Colby formula whichis described hereinafter and generally used (S. R. Colby in Wheeds, 15(1967), 20-22).

[0064] To do this it was initially necessary to calculate the growthinhibition E to be expected theoretically with an additive behavior (theexpected efficacy). This is given by:

E=W1+W2−((W1×W2)/100)

[0065] where W1 and W2 indicate the efficacies of the individualproteins, which is defined as that percentage deviation of the growthplot (in the presence of the protein) from the untreated control. Theefficacy for a protein (at a defined time in the growth plot) is givenby:

W1=(OD(K)−OD(P))/OD(K)×100 (percent)

[0066] In this, OD(K) is the optical density of the untreated controland OD(P) is the optical density of the culture treated with theprotein.

[0067] Thus, on combined use of two proteins, the following statementswere possible: if the efficacy G measured in the experiment is identicalto the expected value E, the behavior is additive. If, on the otherhand, G is greater than E, the behavior is synergistic.

[0068] Using this test model, it emerged that the proteins ChiS, PSI,AFP, ChiG and GluG used in the Example surprisingly have synergisticinhibitory effects on various fungi, and these effects were achievedboth by the combination of two types of protein and by multiplecombination of the abovementioned proteins.

[0069] For example, the following values were determined from thecombination of ChiS and PSI protein and from the combination of AFPprotein and PSI protein on the fungus Rhizoctonia solani (in each casetwo different ChiS and AFP concentrations with a constant RIPconcentration):

[0070] ChiS+PSI

[0071] The expected values were: E1=29.9% and E2=44.5% The measuredvalues were: G1=60.4% and G2=64.1% The proteins ChiS and PSI thereforeact together in a synergistic manner in the inhibition of the growth ofR. Solani.

[0072]FIG. 1 shows the results obtained with the combination of theproteins and with the individual substances. According to the Figure,various ChiS concentrations (0.5 μg/ml and 0.05 μg/ml) are combined withPSi protein (1.0 μg/ml).

[0073] AFP+PSI

[0074] The expected values were: E1=39.9% and E2=41.9% The measuredvalues were: G1=57.7% and G2=65.4% The AFP and PSI combination alsoaccording to this shows a synergistic Inhibition of growth of the fungusR. Solani. FIG. 2 indicates the test results with various AFPconcentrations (0.4 μg/ml and 0.04 μg/ml) combined with PSI protein (1.0μg/ml).

EXAMPLE 2

[0075] Transgenic plants

[0076] In order to obtain the organisms according to the invention withDNA sequences which act together synergistically, initially transgenicplants which contained at least one of the genes which act togethersynergistically were generated.

[0077] ChiS in transgenic plants

[0078] Initially a ChiS gene was fused to plant regulatory sequences.

[0079] A ChiS gene 1.8 Kb in size was sequenced by using syntheticoligonucleotides in the dideoxy sequencing method of Sanger et al. inProc. Natl. Acad. Sci. USA, 74 (1977), 5463-5467.

[0080] The 35S promoter originating from cauliflower mosaic virus (CamV)(400 bp (according to Töpfer et al. in Nucl. Acid. Res., 15 (1987),5890)) underwent transcriptional fusion to the ChiS gene. Thetermination signal, which is 0.2 Kb in size, of the 35S gene of CamV,whose functionality in dicotyledonous plants is known, was used 3′ fromthe ChiS gene. The chimeric gene 35S-ChiS was cloned into the pLS034vector by means of the Agrobacterium tumefaciens transformation systemin tobacco and potato plants, and kanamycin-resistant plants wereregenerated.

[0081] It was possible to detect both the ChiS gene and thecorresponding mRNA as well as the gene product protein in the resultingplants.

[0082] PSI in transgenic plants

[0083] PolyA RNA was initially isolated from ripe barley seeds (Horaeumvulgare L. cv. Piggy) and deposited in a cDNA gene bank inλ-gt-11-phages. The details of the process are to be found in R. Lea inPlant. Biol., 12 (1989), 673-682. Monospecific PSI antibodies were thenused to identify cDNA clones.

[0084] Subsequently, the PSI-positive λ-gt-11-phages were isolated,cloned further and sequenced by the dideoxy sequencing method of Sangeret al. indicated above. The DNA cloned into E. coli was then transferredin the manner described above by conjugation into Agrobacterium LBA4404.

[0085] Both the transferred gene and mRNA and gene product weredetectable in corresponding transgenic tobacco, potato, rape, strawberryand tomato plants.

[0086] AFP in transgenic plants

[0087] For the cloning in the vector, the cDNA sequence of theantifungal peptide is provided with ends which can be ligated into BamH1and Sall restriction cleavage sites. The cloning vector used was pDH51(Pietrzak et al. in Nucl. Acids Res. 14 (1986), 5857). The vector pDH51was opened with the restriction enzymes BamH1 and Sall between promoterand terminator. The vector pDH51 is a pUC18 derivative which containspromoter and terminator sequences of the 35S transcript from cauliflowermosaic virus. These sequences are recognized by the plant'stranscription apparatus and lead to strong constitutive expression ofthe gene associated with them in plants. The DNA of the antifungalpeptide is then cloned via the BamH1 and Sall cleavage site into thevector. Finally, the transcription unit—promoter, gene and terminator—iscut out of the vector using the restriction enzyme EcoRI and cloned intoa plant transformation vector. The following vectors and theirderivatives can, for example, be used as plant transformation vector:

[0088] pOCA18 (Olszewski et al. in Nucl. Acids Res., 16 (1988), 10765)pPCV310 (Koncz and Shell in MGG 204 (1986), 383) and pBin19 (Bevan etal. Nucl. Acids. Res. 12 (1984), 8711)

[0089] After the transcription unit and the vector had been ligated viathe EcoRI cleavage site, the construct was conjugated into theAgrobacterium strain MP90RK (Koncz and Shell (see above)) or IHA101(Hood et al. in J. Bacteriol. 168 (1986), 1291).

[0090] Transgenic tobacco, potato, strawberry, rape and tomato plantswere then transformed by the method described above. Transformed shootsare selected on the basis of the cotransferred resistance to theantibiotic kanamycin. Expression of the antifungal protein in thetransformed crop plants was checked and confirmed by DNA analysis(Southern blotting), RNA analysis (Northern blotting) and proteinanalysis with specific antibodies (Western blotting).

[0091] ChiG and GluG in transgenic plants

[0092] ChiG- and GluG-transgenic plants which were both Southern-,Northern- and Western-positive were obtainable in analogy to the plantsdescribed above.

[0093] ChiS, PSI, AFP, ChiG, GluG in transgenic monocotyledonous plants

[0094] It was possible by means of direct gene transfer to integrate theabovementioned genes into the genome of monocotyledonous plants such as,for example, corn. This resulted in transgenic plants which wereSouthern- and Northern- and Western-positive.

[0095] Combination of various fungus-resistance genes in transgenicplants

[0096] The previously obtained tobacco, corn, rape, strawberry, potatoand tomato plants were crossed together and selected for plantscontaining in each case the fungus-resistant genes of both parents. Inaddition, transgenic plants were obtained by transforming them initiallywith one and then with one or more other gene. Finally, plants were alsotransformed with vectors which contained various resistance genes.Fungus-resistance tests were done with this plant material.Surprisingly, in all cases synergistic effects, not just additiveeffects, in respect of fungus resistance are observed.

[0097] For example, a tobacco plant which expresses ChiS and PSI shows aconsiderably greater resistance to Rhizoctonia infestation than theplants which expressed only ChiS or PSI or which would result from theadditive resistance.

[0098] A synergistic inhibitory effect on infestation with Rhizoctoniasolani also results from combined expression of PSI- and AFP-transgenictobacco. Combination of two or more different genes (ChiS, RIP, AFP,ChiG and GluG) in a wide variety of transgenic plants also led tosynergistic inhibitory effects on various fungi.

[0099] Whereas wild-type plants have index values from 38 to 46 in testson 20 seedlings, it emerges with transgenic tobacco according to theinvention that the latter grows as well in the presence of the fungusRhizoctonia solani as do control plants (index value 10-12) cultivatedon Rhizoctonia-free soil.

[0100] Sequence listing A and A′ (AFP)

[0101] Seq IDNo.: 1 (A)

[0102] Sequence type: complete nucleotide sequence with correspondingprotein to the extent that it is encoded by an open reading frame,active protein (A′)

[0103] Sequence length: 51 amino acids (A′)

[0104] Strandedness: single strand

[0105] Topology: linear

[0106] Molecule type: cDNA

[0107] Original source: Aspergillus giganteus fermentation broth

[0108] Name: antifungal peptide (AFP)

[0109] Features (A): open reading frame of 177 nucleotides, theN-terminal amino acid of the active protein is marked by *.

[0110] Properties: antifungal agent, especially on Rhizoctonia solani,various Aspergillus, Fusaria and Trichophyton species.TTGCGACCCCCGTTGAAGCCGATTCTCTCACCGCTGGTGGTCTGGATGCAAGAGATGAGA   1------------------------------------------------------------  60AACGCTGGGGGCAACTTCGGCTAAGAGAGTGGCGACCACCAGACCTACGTTCTCTACTCT                                             M  Q  E  M  4 -GCGCGGCTTTTGGCCACATACAATGGCAAATGCTACAAGAAGGATAATATCTGCAAGTAC  61------------------------------------------------------------ 120CGCGCCCAAAACCGGRGRATGTTACCGTTTACGATGTTCTTCCTATTATAGACGTTCATGA  R  V  L  A  T  Y  N  G  K  C  Y  K  K D N I C K Y -AAGGCACAGAGCGGCAAGACTGCCATTTGCAAGTGCTATGTCAAAAAGTGCCCCCCGGAC 121------------------------------------------------------------ 180TTCCGTGTCTCGCCCTTCTGACGGTAAACGTTCACGATACAGTTTTTCAGGGGGCGGGCTGK  A  Q  S  G  K  T  A  I  C  K  C  Y  V  K  X  C  P  R  D -GGCGGCGAAATGCGAGTTTGACAGCTACAAGGGGAAGTGCTACTGCTAGACGGTGAGCGAA 181------------------------------------------------------------ 240CCGCGCTTTACGCTCAAACTGTCGATGTTCCCCTTCACGATGACGATCTGCCACTCGCGTTG  A  K  C  E  F  D  S  Y  K  G  K  C  Y  C  *GGGACGAATAGGCTGGGGGTTATTTTACTCTGCT 241----------------------------------- 275CCCTGCTTCATCCGACCCCCAATAAAATGAGACGA A′Ala-Thr-Tyr-Asn-Gly-Lys-Cys-Tyr-Lys-Lys-Asp-Asn-Ile-Cys-Lys-Tyr-Lys-Ala-Gln-Ser-Gly-Lys-Thr-Ala-Ile-Cys-Lys-Cys-Tyr-Val-Lys-Lys-Cys-Pro-Arg-Asp-Gly-Ala-Lys-Cys-Glu-Phe-Asp-Ser-Tyr-Lys-Gly-Lys-Cys-Tyr-Cys.

[0111]Ala-Thr-Tyr-Asn-Gly-Lys-Cys-Tyr-Lys-Lys-Asp-Asn-Ile-Cys-Lys-Tyr-Lys-Ala-Gln-Ser-Gly-Lys-Thr-Ala-Ile-Cys-Lys-Cys-Tyr-Val-Lys-Lys-Cys-Pro-Arg-Asp-Gly-Ala-Lys-Cys-Glu-Phe-Asp-Ser-Tyr-Lys-Gly-Lys-Cys-Tyr-Cys.

[0112] Sequence listing B and B′ (PSI)

[0113] Seq IDNo.: 2

[0114] Sequence type: nucleotide with corresponding protein

[0115] Sequence length: 1078 base pairs (B′=incomplete PSI-cDNA clone)

[0116] Strandedness: single strand

[0117] Topology: linear

[0118] Molecule type: complementary DNA

[0119] Original source: barley seeds (Hordeum vulgare L. cv. Piggy)

[0120] Immediate experimental source: cDNA gene bank in λ-gt-11 phages

[0121] Name: protein synthesis inhibitor

[0122] Features: 42 bp-lona 5′-non-translating region open reading frameof 843 base pairs (the stop codon is marked by an asterisk) 193 basepair-long 3′-non-translated end, possible polyadenylation signals areunderlined

[0123] Properties: antifungal activity, especially on spores ofTrichoderma reesii and fusarium sporotrichoides and on Rhizoctoniasolani. CTTAATAGCACATCTTGTCCGTCTTAGCTTTGCATTACATCCATGGCGGCAAAGATGGCG                         *                 M  A  A  K  M  A                                          -1  1AAGAACGTGGACAAGCCGCTCTTCACCGCGACGTTCAACGTCCAGGCCAGCTCCGCCGAC K  N  V  D  K  F  L  F  T  A  T  F  N  V  Q  A  S  S  A  D             10                            20TACGCCACCTTCATCGCCGGCATCCGCAACAAGCTCCGCAACCCGGCGCACTTCTCCCAC Y  A  T  F  I  A  G  I  R  N  K  L  R  N  P  A  H  F  S  H             30                            40AACCGCCCCGTGCTGCCGCCGGTCGAGCCCAACGTCCCGCCGAGCAGGTGGTTCCACGTG N  R  P  V  L  P  P  V  E  P  F  V  P  P  S  R  W  F  H  V             50                            60GTGCTCAAGGCCTCGCCGACCAGCGCCGGGCTCACGCTGGCCATTCGGGCGGACAACATC V  L  K  A  S  P  T  S  A  G  L  T  L  A  I  R  A  D  N  I             70                            80TACCTGGAGGGCTTCAAGAGCAGCGACGGCACCTGGTGGGAGCTCACCCCGGGCCTCATC Y  L  E  G  F  K  S  S  D  G  T  W  W  E  L  T  P  G  L  I             90                           100CCCGGCGCCACCTACGTGGGGTTCGGCGGCACCTACCGCGACCTCCTCGGCGACACCGAC P  G  A  T  Y  V  G  F  G  G  T  Y  R  D  L  L  G  D  T  D            110                            120AAGCTGACCAACGTCGCTCTCGGCCGGCAGCAGCTCCCGGACGCGGTGACCGCCCTCCAC K  L  T  N  V  A  L  G  R  Q  Q  L  A  D  A  V  T  A  L  B            130                           140GGGCGCACCAAGGCCGACAAGCCGTCCGGCCCGAAGCAGCAGCAGGCGAGGGAGGCGGTG G  R  T  K  A  D  K  P  S  G  P  K  Q  Q  Q  A  R  E  A  V            150                           160CCGACGCTGCTGGTCATGCTGAACGAGGCCACGCGGTTCCAGACGGTGTCTGGGTTCGTG T  T  L  L  L  M  V  N  I  A  T  R  T  Q  T  V  S  G  E  V            170                           180GCCGGGTTGCTGCACCCCAAGGCGGTGGAGAAGAAGAGCGGGAAGATCGGCAATGAGATG A  G  L  L  M  P  K  A  V  E  K  K  S  G  K  I  G  N  E  M            190                           200AAGGCCCAGGTGAACGGGTGGCAGGACCTGTCCGCGGCGCTGCTGAAGACGGACGTGAAG K  A  Q  V  N  G  W  Q  D  L  S  A  A  L  L  K  T  D  V  K            210                           220CCTCCGCCGGGAAAGTCGCCAGGGAAGTTCGCGCCGATCGAGAAGATGGGCGTGAGGACG P  P  P  G  K  S  P  A  K  F  A  P  I  E  K  M  G  V  R  T            230                           240GCTGTACAGGCCGCCAACACCCTGGGGATCCTGCTGTTCGTGGAGGTGCCGGGTGGGTTG A  V  Q  A  A  N  T  L  G  I  L  L  F  V  E  V  P  G  G  L            250                           260ACGGTGGCCAAGGCGCTGGACGTGTTCCATGCGAGTGGTGGGAAATAGGTAGTTTTCCAG T  V  A  K  A  L  E  L  F  H  A  S  G  G  K  *GTATACGTGCATGGGTAGTGTAAAAGTCG+E,uns AATAAACATGTCACAGAGTGACGGACTGATATA+E,uns AATAAATAAATAAACGTGTCACAGAGTTACATATAAACA+E,unsAATAAATAAATAATTAAAA ATGTCCAGTTTA₄₇TCGGTGACGACGCTGCTCCTCATGGTGAACGAGGCCACGCGGTTCCAGACGGTGTCGGGG A  V  T  T  L  L  L  M  V  N  E  A  T  R  F  Q  T  V  S  G                  170                           180TTCGTGGCCGGGCTGCTGCACCCCAAGGCGGTGGAGAAGAAGAGCGGGAAGATCGGCAAT F  V  A  G  L  L  H  P  K  A  V  E  K  K  S  G  K  I  G  N                  190                           200GAGATGAAGGCCCAGGTGAACGGGTGGCAGGACCTGTCCGCGCGGCTGCTGAAGACGGAC E  M  K  A  Q  V  N  G  N  Q  D  L  S  A  A  L  L  K  T  D            210                           220GTGAAGCCCCCGCCGGGAAAGTCGCCAGCGAAGTTCACGCCGATCGAGAAGATGGGCGTG V  K  P  P  P  G  K  S  P  A  K  F  T  P  I  E  K  M  G  V                  230                           240AGGACTGCTGAGCAGGCTGCGGCTACTTTGGGGATCCTGCTGTTCGTTGAGGTGCCGGGT R  T  A  E  Q  A  A  A  T  L  G  I  L  L  F  V  E  V  P  G                  250                           260GGGTTGACGGTGGCCAAGGCGCTGGAGCTGTTTCATGCGAGTGGTGGGAAATAGGTAGTT G  L  T  V  A  K  A  L  E  L  F  H  A  S  G  G  K  *            270                           280TTGCAGGTATACCTGCATGGGTAAATGTAAAAGTCG+E,uns AATAAAAATGTCACAGAGTGACGGACTGATATA+E,uns AATAAATT+E,unsAATAAACATGTCATCATGAGTGACAGACTGATATAAATAAATA

[0124] Sequence listing C (ChiS)

[0125] Seq IDNo.: 3

[0126] Sequence type: nucleotide

[0127] Strandedness: single strand (the activated strand is doublestrand)

[0128] Topology: linear

[0129] Molecule type: cDNA

[0130] Immediate experimental source: plasmid pLChiS from E. Coli strainA 5187

[0131] Original source: Cosmid bank from Serratia Marcescens

[0132] Name: ChiS protein (chitinase)

[0133] Properties: exo-chitinase    1 CAGGGCGTTG TCAATAATGA CAACACCCTGGCTGAAGAGT GTGGTGCAAT   51 ACTGATAAAT ATTTATCTTT GGTTAATAGA AAATTCACTATCCTTATTTG  101 TCATGTTTTG ATTTATCTTT GCTTAATAGA ATTCACGCTT GCTGAATAAA 151 ACGCAGTTGA TAGCGCTGTT GTTTTTGCGC CTTTTTTATT TATAGTACTG  201AATGTACGCG GTGGGAATGA TTATTTCGCC ACGTGGAAAG ACGCTGTTGT  251 TATTTATTGATTTTAACGTT CGCGGATTAT TGCGGAATTT TTTCGCTTCG  301 GCAATGCATC GCGACGATTAACTCTTTTAT GTTTATCCTC TCGGAATAAA  351 GGAATCAGTT ATGCGCAAAT TTAATAAACCGCTGTTGGCG CTGTTGATCG  401 GCAGCACGGT GTGTTCCGCG GCGCAGGCCG CCGCGCCGGGCAAGCCGACG  451 ATCGCCTGGG GCAACACCAA GTTCGCCATC GTTGAAGTTG ACCAGGCGGC 501 TACCGCTTAT AATAATTTGG TGAAGGTAAA AAATGCCGCC GATGTTTCCG  551TCTCCTGGAA TTTATGGAAT GGCGACACCG GCACGACGGC AAAAGTTTTA  601 TTAAATGGCAAAGAGGCGTG GAGTGGTCCT TCAACCGGAT CTTCCGGTAC  651 GGCGAATTTT AAAGTGAATAAAGGCGGCGG TTATCAAATG CAGGTGGCAC  701 TGTGCAATGC GGACGGCTGC ACCGCCAGTGACGCCACCGA AATTGTGGTA  751 GCCGACACCG ACGGCAGCGA TTTGGCGCCG TTGAAAGAGCCGCTGCTGGA  801 AAAGAATAAA CGGTATAAAC AGAACTCCGG CAAAGTGGTC GGTTCTTATT 851 TCGTCGAGTG GGGCGTTTAC GGGCGCAATT TCACCGTCGA CAAGATCCCG  901GCGCAAAACC TGACCCACCT GCTGTACGGC TTTATCCCGA TCTGCGGCGG  951 CAATGGCATCAACGACAGCC TGAAAGAGAT TGAAGGCAGC TTCCAGGCGT 1001 TGCAGCGCTC CTGCCAGGGCCGCGAGGACT TCAAAGTCTC GATCCACGAT 1051 CCGTTCGCCC CGCTGCAAAA AGCGCAGAAGGGCGTGACCG CCTGGGATGA 1101 CCGGTACAAG GGCAACTTCG GCCAGCTGAT GGCGCTGAAGCAGGCGCATC 1151 CTGACCTGAA AATCCTGCCG TCGATCGGCG GCTGGACGCT GTCCGACCCG1201 TTCTTCTTCA TGGGCGACAA GGTGAAGCGC GATCGCTTGG TCGGTTCGGT 1251GAAAGAGTTC CTGCAGACCT GGAAGTTCTT CGACGGCGTG GATATCGACT 1301 GGGAGTTGGGGGGCGGCAAA GGCGCCAACC CTAACCTGGG CAGCCCGCAA 1351 GACGGGGAAA CGTATGTGCTGCTGATGAAG GAGCTGCGGG CGATGCTGGA 1401 TCAGCTGTCG GTGGAAACGG GCCGCAAGTATGAGCTGACC TCCGCCATCA 1451 GCGCCGGTAA GGACAAGATC GACAAGGTGG CTTACAACGTTGCGCAGAAC 1501 TCGATGGATC ACATGTTGCT GATGAGCTAC GACTTCTATG GCGCCTTCGA1551 TCTGAAGAAC GTGGGGCATC AGACCGCGCT GAATGCGCCG GCCTGGAAAC 1601CGGACACCGC CTACACCACG GTGAACGGCG TCAATGCGCT GCTGGCGCAG 1651 GGCGTCAAGCCGGGCAAAAT CGTCGTCGGC ACCGCCATGT ATGGCCGCGG 1701 CTGGACCGGG GTGAACGGCTACCAGAACAA TATTCCGTTC ACCGGCACCG 1751 CCACCGGGCC GGTTAAAGGC ACCTGGGAGAACGGTATCGT GGACTACCGC 1801 CAAATCGCCG GCCAGTTCAT GAGCGGCGAG TGGCAGTATACCTACGACGC 1851 CACGGCGGAA GCGCCTTACG TGTTCAAACC TTCCACCGGC GATCTGATCA1901 CCTTCGACGA TGCCCGCTCG GTGCAGGCTA AAGGCAAGTA CGTGTTGGAT 1951AAGCAGCTGG GCGGCCTGTT CTGGTGGGAG ATCGACGCGG ATAACGGCGA 2001 TATTCTCAACAGCATGAACG CCAGCCTGGG CAACAGCGCC GGCGTTCAAT 2051 AATCGGTTGC AGTGGTTGCCGGGGGATATC CTTTCGCCCC CGGCTTTTTC 2101 GCCGACGAAA GTTTTTTTAC GCCGCACAGATTGTGGCTCT GCCCCGAGCA 2151 AAACGCGCTC ATCGGACTCA CCCTTTTGGG TAATCCTTCAGCATTTCCTC 2201 CTGTCTTTAA CGGCGATCAC AAAAATAACC GTTCAGATAT TCATCATTCA2251 GCAACAAAGT TTTGGCGTTT TTTAACGGAG TTAAAAACCA GTAAGTTTGT

[0134] Sequence listing D (ChiG)

[0135] Seq IDNo.: 4

[0136] Sequence type: nucleotide

[0137] Sequence length: 1013 nucleotides

[0138] Molecule type: cDNA

[0139] Original source: barley seeds (Hordeum vulgare L.)

[0140] Name: ChiG (chitinase G)

[0141] Feature: 63 pb-long 5′-non-translating initial region, 798 pbopen reading frame, 152 pb-long 3′-non-translated end, reading stopcodons are marked by an asterisk, the probable signal peptide sequencesare underlined, the amino-acid sequence of a 26 kD chitinase preproteinwith 266 amino acids is indicated below the nucleotide sequence, theunderlined AT-rich sequence in position 905 is probably apolyadenylation signal.

[0142] Properties antifungal activity, especially on Trichoderma reesiiand Fusarium sporotrichoides as well as Rhizoctonia solani and Botrytiscinerea. D CCTACGACAGTAGCGTAACGGTAAACACCGAGTACGGTACTCTGTGCTTTGTTGGCTCGC  60                 *     *ACAATGAGATCGCTCGCGGTGGTGGTGGCCGTGGTAGCCACGGTGGCCATGGCCATCGGC  120   +E,uns  M  R  S  L  A  V  V  V  A  V  V  A  T  V  A  M  A  I  G             −20                           −10ACGGCGCGCGGCAGCGTGTCCTCCATCGTCTCGCGCGCACAGTTTGACCGCATGCTTCTC  180 +E,uns T  A  R  G  S  V  S  S  I  V  S  R  A  Q  F  D  R  M  L  L         −1  1                           10CACCGCAACGACGGCGCCTGCCAGGCCAAGGGCTTCTACACCTACGACGCCTTCGTCGCC  240 +E,unsR  R  N  D  G  A  C  Q  A  K  G  F  Y  T  Y  D  A  F  V  A          20                            30GCCGCAGCCGCCTTCCCGGGCTTCGGCACCACCGGCAGCGCCGACGCCCAGAAGCGCGAG  300 +E,uns A  A  A  A  F  P  G  F  G  T  T  G  S  A  D  A  Q  K  R +E,uns  E           40                            50GTGGCCGCCTTCCTAGCACAGACCTCCCACGAGACCACCGGCGGGTGGGCGACTGCACCG  360 +E,uns V  A  A  F  L  A  Q  T  S  H  E  T  T  G  G  W +E,uns  A  T  A  P           60                            70GACGGGGCCTTCGCCTGGGGCTACTGCTTCAAGCAGGAACGTGGCGCCTCCTCCGACTAC  420 +E,uns D  G  A  F  W  W  G  Y  C  F  K  Q  E  R  G  A  S  S  D  Y          80                             90TGCACCCCGAGCGCACAATGGCCGTGCGCCCCCGGGAAGCGCTACTACGGCCGCGGGCCA  480 C  T  P  S  A  Q  W  P  C  A  P  G  K  R  Y +E,uns  Y  G  R  G  P          100                           110ATCCAGCTCTCCCACAACTACAACTATGGACCTGCCGGCCGGGCCATCGGGGTCGATCTG  540 +E,uns I  Q  L  S  H  N  Y  N  Y  G  P  A  G  R  A  I  G  V  D  L          120                           130CTGGCCAACCCGGACCTGGTGGCCACGGACGCCACTGTGGGCTTTAAGACGGCCATCTGG  600 +E,uns L  A  N  P  D  L  V  A  T  D  A  T  V  G  F  K  T  A  I  W         140                           150TTCTGGATGACGGCGCAGCCGCCCAAGCCATCGAGCCATGCTGTGATCGCCGGCCAGTGG  660 F  W +E,uns  M  T  A  Q  P  P  K  P  S  S  H  A  V  I  A  G  Q  W          160                           170AGCCCGTCAGGGGCTGACCGGGCCGCAGGCCGGGTGCCCGGGTTTGGTGTGATCACCAAC  720 +E,uns S  P  S  G  A  D  R  A  A  G  R  V  P  G  F  G  V  I  T  N          180                           190ATCATCAACGGCGGGATCGAGTGCGGTCACGGGCAGGACAGCCGCGTCGCCGATCGAATC  780 +E,uns I  I  N  G  G  I  E  C  G  H  G  Q  D  S  R  V  A  D  R  I          200                           210GGGTTTTACAAGCGCTACTGTGACATCCTCGGCGTTGGCTACGGCAACAACCTCGATTGC  840 +E,uns G  F  Y  K  R  Y  C  D  I  L  G  V  G  Y  G  N  N  L  D  C          220                           230TACAGCCAGAGACCCTTCGCCTAATTAATTAGTCATGTATTAATCTTGGCCCTCCATAAA  900 +E,uns Y  S  Q  R  P  F  A  *          240 ATAC+E,unsAATAAGAGCATCGTCTCCTATCTACATGCTGTAAGATGTAACTATGGTAACCTTTT  960ATGGGGAACATAACAAAGGCATCTCGTATAGATGCTTTGCTA₁₂ 1013

[0143] Sequence listing E (GluG)

[0144] Seq IDNo.: 5

[0145] Sequence type: nucleotide with corresponding protein

[0146] Sequence length: 1249 nucleotides

[0147] Molecule type: cDNA

[0148] Original source: barley seed (Hordeum vulgare L.)

[0149] Name: GluG (glucanase)

[0150] Feature: 48 bp-long 5′-non-translating initial region openreading frame of 1002 bp 199 pb-long 3′-non-translated end, theunderlined At-rich sequence at position 1083 and 1210 are probablypolyadenylation signals, the derived amino-acid sequence of the encodedpreprotein of 334 amino acids is indicated below the nucleotidesequence. E GGCAGCATTGCATAGCATTTGAGCACCAGATACTCCGTGTGTGCACCAATGGCTAGAAAA  60                                                 +E,uns  M  A  R  K                                                 −28                      *                  *GATGTTGCCTCCATGTTTGCAGTTGCTCTCTTCATTGGAGCATTCGCTGCTGTTCCTACG  120 +E,uns D  V  A  S  M  F  A  V  A +E,uns    L  I  G  A  F  A  A  V  P  T             −20                           −10AGTGTGCAGTCCATCGGCGTATGCTACGGCGTGATCGGCAACAACCTCCCCTCCCGGAGC  180 +E,uns S  V  Q  S  I  G  V  C  Y  G  V  I  G  N  N  L  P  S  R  S          −1 +1                         10GACGTGGTGCAGCTCTACAGGTCCAAGGGCATCAACGGCATGCGCAT{overscore(CTACTTCGCCGAC)}  240 D  V  V  Q  L  Y  R  S  K  G  I  N  G  M  R  I  Y  F  A  D          20                            30 {overscore(GGGCAGGCCCTCTCGGCCGTCCGCAACTCCGGCATCGGCCTCATCCTCGACATCGGC)}AAC  300 G  Q  A  L  S  A  V  R  N  S  G  I  G  L  I  L  D  I  G  N          40                            50GACCAGCTCGCCAACATCGCCGCCAGCACCTCCAACGCGGCCTCCTGGGTCCAGAACAAC  360 D  Q  L  A  N  I  A  A  S  T  S  N  A  A  S  W  V  Q  N  N          60                            70GTGCGGCCCTACTACCCTGCCGTGAACATCAAGTACATCGCCGCCGGCAACGAGGTGCAG  420 V  R  P  Y  Y  P  A  V  N  I  K  Y  I  A  A  G  N  E  V  Q          80                            90GGCGGCGCCACGCAGAGCATCCTGCCGGCCATGCGCAACCTCAACGCGGCCCTCTCCGCG  480 G  G  A  T  Q  S  I  L  P  A  M  R  N  L  N  A  A  L  S  A         100                           110GCGGGGCTCGGCGCCATCAAGGTGTCCACCTCCATCCGGTTCGACGAGGTGGCCAACTCC  540 A  G  L  G  A  I  K  V  S  T  S  I  R  F  D  E  V  A  N  S         120                           130TTCCCGCCCTCCGCCGGCGTGTTCAAGAACGCCTACATGACGGACGTGGCCCGGCTCCTG  600 F  P  P  S  A  G  V  F  K  N  A  Y  M  T  D  V  A  R  L  L         140                           150GCGAGCACCGGCGCGCCGCTGCTCGCCAACGTCTACCCCTACTTCGCGTACCGTGACAAC  660 A  S  T  G  A  P  L  L  A  N  V  Y  P  Y  F  A  Y  R  D  N         160                           170CCCGGGAGCATCAGCCTGAACTACGCGACGTTCCAGCCGGGCACCACCGTGCGTGACCAG  720 P  G  S  I  S  L  N  Y  A  T  F  Q  P  G  T  T  V  R  D  Q         180                           190AACAACGGGCTGACCTACACGTCCCTGTTCGACGCGATGGTGGACGCCGTGTACGCGGCG  780 N  N  G  L  T  Y  T  S  L  F  D  A  M  V  D  A  V  Y  A  A         200                           210CTGGAGAAGGCCGGCGCGCCGGCGGTGAAGGTGGTGGTGTCGGAGAGCGGGTGGCCGTGG  840 L  E  K  A  G  A  P  A  V  K  V  V  V  S  E  S  G  W  P  S         220                           230GCGGGCGGGTTTGCGGCGTCGGCCGGCAATGCGCGGACGTACAACCAGGGGCTGATCAAC  900 A  G  G  F  A  A  S  A  G  N  A  R  T  Y  N  Q  G  L  I  N         240                           250CACGTCGGCGGGGGCACGCCCAAGAAGCGGGAGGCGCTGGAGACGTACATCTTCGCCATG  960 H  V  G  G  G  T  P  K  K  R  E  A  L  E  T  Y  I  F  A  M         260                           270TTCAAGGAGAACCAGAAGACCGGCCACGCCACGGAGAGGAGCTTCGGGCTCTTCAACCCG 1020 F  N  E  N  Q  K  Y  G  D  A  T  E  R  S  F  G  L  F  N  P         280                           290GACAAGTCGCCGGCATACAACATCCAGTTCTAGTACGTGTAGCTACCTAGCTCACATACC 1080 D  K  S  P  A  Y  N  I  Q  F  *          300 TA+E,unsAATAAATAAGCTGCACGTACGTACGTAATGCGGCATCCAAGTGTAACGTAGACACGTA 1140CATTCATCCATGGAAGAGTGCAACCAAGCATGCGTTAACTTCCTGGTGATGATACATCAT 1200CATGGTATGAATAAAAGATATGGAAGATGTTATGA₁₅ 1249

[0151]

1 12 275 base pairs nucleic acid single linear cDNA Aspergillusgiganteus 5′UTR 1..45 CDS 46..225 experimental /codon_start= 46/function=“antifungal agent” /product=“antifungal peptide)”/evidence=EXPERIMENTAL /note= “antifungal agent, especially onRhizoctonia solani, various Aspergillus, Fusaria and Trichophytonspecies” 1 TTGCCACCCC CGTTGAAGCC GATTCTCTCA CCGCTGGTGG TCTGG ATG CAA GAG54 Met Gln Glu 1 ATG AGA GCG CGG GTT TTG GCC ACA TAC AAT GGC AAA TGC TACAAG AAG 102 Met Arg Ala Arg Val Leu Ala Thr Tyr Asn Gly Lys Cys Tyr LysLys 5 10 15 GAT AAT ATC TGC AAG TAC AAG GCA CAG AGC GGC AAG ACT GCC ATTTGC 150 Asp Asn Ile Cys Lys Tyr Lys Ala Gln Ser Gly Lys Thr Ala Ile Cys20 25 30 35 AAG TGC TAT GTC AAA AAG TGC CCC CGC GAC GGC GCG AAA TGC GAGTTT 198 Lys Cys Tyr Val Lys Lys Cys Pro Arg Asp Gly Ala Lys Cys Glu Phe40 45 50 GAC AGC TAC AAG GGG AAG TGC TAC TGC TAGACGGTGA GCGAAGGGAC 245Asp Ser Tyr Lys Gly Lys Cys Tyr Cys 55 60 GAAGTAGGCT GGGGGTTATTTTACTCTGCT 275 60 amino acids amino acid linear protein not provided 2Met Gln Glu Met Arg Ala Arg Val Leu Ala Thr Tyr Asn Gly Lys Cys 1 5 1015 Tyr Lys Lys Asp Asn Ile Cys Lys Tyr Lys Ala Gln Ser Gly Lys Thr 20 2530 Ala Ile Cys Lys Cys Tyr Val Lys Lys Cys Pro Arg Asp Gly Ala Lys 35 4045 Cys Glu Phe Asp Ser Tyr Lys Gly Lys Cys Tyr Cys 50 55 60 51 aminoacids amino acid linear protein C-terminal Aspergillus giganteus Protein1..51 /note= “active protein fragment of AFP” 3 Ala Thr Tyr Asn Gly LysCys Tyr Lys Lys Asp Asn Ile Cys Lys Tyr 1 5 10 15 Lys Ala Gln Ser GlyLys Thr Ala Ile Cys Lys Cys Tyr Val Lys Lys 20 25 30 Cys Pro Arg Asp GlyAla Lys Cys Glu Phe Asp Ser Tyr Lys Gly Lys 35 40 45 Cys Tyr Cys 50 1032base pairs nucleic acid single linear cDNA Hordeum vulgare L.cv. PiggycDNA gene bank in lambda-gt-11-phages 5′UTR 1..42 CDS 43..885/codon_start= 43 /function=“antifungal activity” /product=“proteinsynthesis inhibitor (PSI)” /note= “antifungal activity, especially onspores of Trichoderma reesii and Fusarium sporotrichoides and onRhizoctonia solani.” 3′UTR 886..1032 /partial /note= “46 nucleotides atthe 3′-end not shown.” polyA_signal 930..935 /note= “potentialpolyadenylation signal” polyA_signal 963..976 /note= “potentialpolyadenylation signal” polyA_signal 1002..1011 /note= “potentialpolyadenylation signal” mat_peptide 46..886 4 CTTAATAGCA CATCTTGTCCGTCTTAGCTT TGCATTACAT CC ATG GCG GCA AAG 54 Met Ala Ala Lys 1 ATG GCGAAG AAC GTG GAC AAG CCG CTC TTC ACC GCG ACG TTC AAC GTC 102 Met Ala LysAsn Val Asp Lys Pro Leu Phe Thr Ala Thr Phe Asn Val 5 10 15 20 CAG GCCAGC TCC GCC GAC TAC GCC ACC TTC ATC GCC GGC ATC CGC AAC 150 Gln Ala SerSer Ala Asp Tyr Ala Thr Phe Ile Ala Gly Ile Arg Asn 25 30 35 AAG CTC CGCAAC CCG GCG CAC TTC TCC CAC AAC CGC CCC GTG CTG CCG 198 Lys Leu Arg AsnPro Ala His Phe Ser His Asn Arg Pro Val Leu Pro 40 45 50 CCG GTC GAG CCCAAC GTC CCG CCG AGC AGG TGG TTC CAC GTC GTG CTC 246 Pro Val Glu Pro AsnVal Pro Pro Ser Arg Trp Phe His Val Val Leu 55 60 65 AAG GCC TCG CCG ACCAGC GCC GGG CTC ACG CTG GCC ATT CGG GCG GAC 294 Lys Ala Ser Pro Thr SerAla Gly Leu Thr Leu Ala Ile Arg Ala Asp 70 75 80 AAC ATC TAC CTG GAG GGCTTC AAG AGC AGC GAC GGC ACC TGG TGG GAG 342 Asn Ile Tyr Leu Glu Gly PheLys Ser Ser Asp Gly Thr Trp Trp Glu 85 90 95 100 CTC ACC CCG GGC CTC ATCCCC GGC GCC ACC TAC GTC GGG TTC GGC GGC 390 Leu Thr Pro Gly Leu Ile ProGly Ala Thr Tyr Val Gly Phe Gly Gly 105 110 115 ACC TAC CGC GAC CTC CTCGGC GAC ACC GAC AAG CTG ACC AAC GTC GCT 438 Thr Tyr Arg Asp Leu Leu GlyAsp Thr Asp Lys Leu Thr Asn Val Ala 120 125 130 CTC GGC CGG CAG CAG CTGGCG GAC GCG GTG ACC GCC CTC CAC GGG CGC 486 Leu Gly Arg Gln Gln Leu AlaAsp Ala Val Thr Ala Leu His Gly Arg 135 140 145 ACC AAG GCC GAC AAG CCGTCC GGC CCG AAG CAG CAG CAG GCG AGG GAG 534 Thr Lys Ala Asp Lys Pro SerGly Pro Lys Gln Gln Gln Ala Arg Glu 150 155 160 GCG GTG ACG ACG CTG CTCCTC ATG GTG AAC GAG GCC ACG CGG TTC CAG 582 Ala Val Thr Thr Leu Leu LeuMet Val Asn Glu Ala Thr Arg Phe Gln 165 170 175 180 ACG GTG TCT GGG TTCGTG GCC GGG TTG CTG CAC CCC AAG GCG GTG GAG 630 Thr Val Ser Gly Phe ValAla Gly Leu Leu His Pro Lys Ala Val Glu 185 190 195 AAG AAG AGC GGG AAGATC GGC AAT GAG ATG AAG GCC CAG GTG AAC GGG 678 Lys Lys Ser Gly Lys IleGly Asn Glu Met Lys Ala Gln Val Asn Gly 200 205 210 TGG CAG GAC CTG TCCGCG GCG CTG CTG AAG ACG GAC GTG AAG CCT CCG 726 Trp Gln Asp Leu Ser AlaAla Leu Leu Lys Thr Asp Val Lys Pro Pro 215 220 225 CCG GGA AAG TCG CCAGCG AAG TTC GCG CCG ATC GAG AAG ATG GGC GTG 774 Pro Gly Lys Ser Pro AlaLys Phe Ala Pro Ile Glu Lys Met Gly Val 230 235 240 AGG ACG GCT GTA CAGGCC GCC AAC ACG CTG GGG ATC CTG CTG TTC GTG 822 Arg Thr Ala Val Gln AlaAla Asn Thr Leu Gly Ile Leu Leu Phe Val 245 250 255 260 GAG GTG CCG GGTGGG TTG ACG GTG GCC AAG GCG CTG GAG CTG TTC CAT 870 Glu Val Pro Gly GlyLeu Thr Val Ala Lys Ala Leu Glu Leu Phe His 265 270 275 GCG AGT GGT GGGAAA TAGGTAGTTT TCCAGGTATA CCTGCATGGG TAGTGTAAAA 925 Ala Ser Gly Gly Lys280 GTCGAATAAA CATGTCACAG AGTGACGGAC TGATATAAAT AAATAAATAA ACGTGTCACA985 GAGTTACATA TAAACAAATA AATAAATAAT TAAAAATGTC CAGTTTA 1032 281 aminoacids amino acid linear protein not provided 5 Met Ala Ala Lys Met AlaLys Asn Val Asp Lys Pro Leu Phe Thr Ala 1 5 10 15 Thr Phe Asn Val GlnAla Ser Ser Ala Asp Tyr Ala Thr Phe Ile Ala 20 25 30 Gly Ile Arg Asn LysLeu Arg Asn Pro Ala His Phe Ser His Asn Arg 35 40 45 Pro Val Leu Pro ProVal Glu Pro Asn Val Pro Pro Ser Arg Trp Phe 50 55 60 His Val Val Leu LysAla Ser Pro Thr Ser Ala Gly Leu Thr Leu Ala 65 70 75 80 Ile Arg Ala AspAsn Ile Tyr Leu Glu Gly Phe Lys Ser Ser Asp Gly 85 90 95 Thr Trp Trp GluLeu Thr Pro Gly Leu Ile Pro Gly Ala Thr Tyr Val 100 105 110 Gly Phe GlyGly Thr Tyr Arg Asp Leu Leu Gly Asp Thr Asp Lys Leu 115 120 125 Thr AsnVal Ala Leu Gly Arg Gln Gln Leu Ala Asp Ala Val Thr Ala 130 135 140 LeuHis Gly Arg Thr Lys Ala Asp Lys Pro Ser Gly Pro Lys Gln Gln 145 150 155160 Gln Ala Arg Glu Ala Val Thr Thr Leu Leu Leu Met Val Asn Glu Ala 165170 175 Thr Arg Phe Gln Thr Val Ser Gly Phe Val Ala Gly Leu Leu His Pro180 185 190 Lys Ala Val Glu Lys Lys Ser Gly Lys Ile Gly Asn Glu Met LysAla 195 200 205 Gln Val Asn Gly Trp Gln Asp Leu Ser Ala Ala Leu Leu LysThr Asp 210 215 220 Val Lys Pro Pro Pro Gly Lys Ser Pro Ala Lys Phe AlaPro Ile Glu 225 230 235 240 Lys Met Gly Val Arg Thr Ala Val Gln Ala AlaAsn Thr Leu Gly Ile 245 250 255 Leu Leu Phe Val Glu Val Pro Gly Gly LeuThr Val Ala Lys Ala Leu 260 265 270 Glu Leu Phe His Ala Ser Gly Gly Lys480 base pairs nucleic acid single linear cDNA Hordeum vulgare L.cv.Piggy cDNA gene bank in lambda-gt-11-phages incomplete psi cDNA cloneCDS 1..351 /partial /codon_start= 1 /function=“protein synthesisinhibitor” /product=“protein synthesis inhibitor” /standard_name= “PSI”/note= “aminoterminally incomplete protein from an incomplete PSI cDNAclone” 3′UTR 352..487 polyA_signal 404..409 /note= “potentialpolyadenylation signal” polyA_signal 437..442 /note= “potentialpolyadenylation signal” polyA_signal 445..450 /note= “potentialpolyadenylation signal” 6 GCG GTG ACG ACG CTG CTC CTC ATG GTG AAC GAGGCC ACG CGG TTC CAG 48 Ala Val Thr Thr Leu Leu Leu Met Val Asn Glu AlaThr Arg Phe Gln 1 5 10 15 ACG GTG TCG GGG TTC GTG GCC GGG CTG CTG CACCCC AAG GCG GTG GAG 96 Thr Val Ser Gly Phe Val Ala Gly Leu Leu His ProLys Ala Val Glu 20 25 30 AAG AAG AGC GGG AAG ATC GGC AAT GAG ATG AAG GCCCAG GTG AAC GGG 144 Lys Lys Ser Gly Lys Ile Gly Asn Glu Met Lys Ala GlnVal Asn Gly 35 40 45 TGG CAG GAC CTG TCC GCG GCG CTG CTG AAG ACG GAC GTGAAG CCC CCG 192 Trp Gln Asp Leu Ser Ala Ala Leu Leu Lys Thr Asp Val LysPro Pro 50 55 60 CCG GGA AAG TCG CCA GCG AAG TTC ACG CCG ATC GAG AAG ATGGGC GTG 240 Pro Gly Lys Ser Pro Ala Lys Phe Thr Pro Ile Glu Lys Met GlyVal 65 70 75 80 AGG ACT GCT GAG CAG GCT GCG GCT ACT TTG GGG ATC CTG CTGTTC GTT 288 Arg Thr Ala Glu Gln Ala Ala Ala Thr Leu Gly Ile Leu Leu PheVal 85 90 95 GAG GTG CCG GGT GGG TTG ACG GTG GCC AAG GCG CTG GAG CTG TTTCAT 336 Glu Val Pro Gly Gly Leu Thr Val Ala Lys Ala Leu Glu Leu Phe His100 105 110 GCG AGT GGT GGG AAA TAGGTAGTTT TGCAGGTATA CCTGCATGGGTAAATGTAAA 391 Ala Ser Gly Gly Lys 115 AGTCGAATAA AAATGTCACA GAGTGACGGACTGATATAAA TAAATTAATA AACATGTCAT 451 CATGAGTGAC AGACTGATAT AAATAAATA 480117 amino acids amino acid linear protein not provided 7 Ala Val Thr ThrLeu Leu Leu Met Val Asn Glu Ala Thr Arg Phe Gln 1 5 10 15 Thr Val SerGly Phe Val Ala Gly Leu Leu His Pro Lys Ala Val Glu 20 25 30 Lys Lys SerGly Lys Ile Gly Asn Glu Met Lys Ala Gln Val Asn Gly 35 40 45 Trp Gln AspLeu Ser Ala Ala Leu Leu Lys Thr Asp Val Lys Pro Pro 50 55 60 Pro Gly LysSer Pro Ala Lys Phe Thr Pro Ile Glu Lys Met Gly Val 65 70 75 80 Arg ThrAla Glu Gln Ala Ala Ala Thr Leu Gly Ile Leu Leu Phe Val 85 90 95 Glu ValPro Gly Gly Leu Thr Val Ala Lys Ala Leu Glu Leu Phe His 100 105 110 AlaSer Gly Gly Lys 115 2329 base pairs nucleic acid single linear cDNASerratia marcescens Cosmid bank from Serratia marcescens misc_feature1..2329 experimental /function= “exo-chitinase” /product=“ChiS protein”/evidence=EXPERIMENTAL /note= “sequence listing of the ChiS gene from aplasmid pLChiS from E.coli A 5187” 8 CAGGGCGTTG TCAATAATGA CAACACCCTGGCTGAAGAGT GTGGTGCAAT ACTGATAAAT 60 ATTTATCTTT CCTTAATAGA AAATTCACTATCCTTATTTG TCATGTTTTC TTTTATTTAT 120 ATGAAAATAA ATTCACGCTT GCTGAATAAAACCCAGTTGA TAGCGCTCTT GTTTTTGCGC 180 CTTTTTTATT TATAGTACTG AATGTACGCGGTGGGAATGA TTATTTCGCC ACGTGGAAAG 240 ACGCTGTTGT TATTTATTGA TTTTAACCTTCGCGGATTAT TGCGGAATTT TTTCGCTTCG 300 GCAATGCATC GCGACGATTA ACTCTTTTATGTTTATCCTC TCGGAATAAA GGAATCAGTT 360 ATGCGCAAAT TTAATAAACC GCTGTTGGCGCTGTTGATCG GCAGCACGCT GTGTTCCGCG 420 GCGCAGGCCG CCGCGCCGGG CAAGCCGACCATCGCCTGGG GCAACACCAA GTTCGCCATC 480 GTTGAAGTTG ACCAGGCGGC TACCGCTTATAATAATTTGG TGAAGGTAAA AAATGCCGCC 540 GATGTTTCCG TCTCCTGGAA TTTATGGAATGGCGACACCG GCACGACGGC AAAAGTTTTA 600 TTAAATGGCA AAGAGGCGTG GAGTGGTCCTTCAACCGGAT CTTCCGGTAC GGCGAATTTT 660 AAAGTGAATA AAGGCGGCCG TTATCAAATGCAGGTGGCAC TGTGCAATGC CGACGGCTGC 720 ACCGCCAGTG ACGCCACCGA AATTGTGGTAGCCGACACCG ACGGCAGCCA TTTGGCGCCG 780 TTGAAAGAGC CGCTGCTGGA AAAGAATAAACCGTATAAAC AGAACTCCGG CAAAGTGGTC 840 GGTTCTTATT TCGTCGAGTG GGGCGTTTACGGGCGCAATT TCACCGTCGA CAAGATCCCG 900 GCGCAAAACC TGACCCACCT GCTGTACGGCTTTATCCCGA TCTGCGGCGG CAATGGCATC 960 AACGACAGCC TGAAAGAGAT TGAAGGCAGCTTCCAGGCGT TGCAGCGCTC CTGCCAGGGC 1020 CGCGAGGACT TCAAAGTCTC GATCCACGATCCGTTCGCCC CGCTGCAAAA AGCGCAGAAG 1080 GGCGTGACCG CCTGGGATGA CCCCTACAAGGGCAACTTCG GCCAGCTGAT GGCGCTGAAG 1140 CAGGCGCATC CTGACCTGAA AATCCTGCCGTCGATCGGCG GCTGGACGCT GTCCGACCCG 1200 TTCTTCTTCA TGGGCGACAA GGTGAAGCGCGATCGCTTCG TCGGTTCGGT GAAAGAGTTC 1260 CTGCAGACCT GGAAGTTCTT CGACGGCGTGGATATCGACT GGGAGTTCCC GGGCGGCAAA 1320 GGCGCCAACC CTAACCTGGG CAGCCCGCAAGACGGGGAAA CCTATGTGCT GCTGATGAAG 1380 GAGCTGCGGG CGATGCTGGA TCAGCTGTCGGTGGAAACCG GCCGCAAGTA TGAGCTGACC 1440 TCCGCCATCA GCGCCGGTAA GGACAAGATCGACAAGGTGG CTTACAACGT TGCGCAGAAC 1500 TCGATGGATC ACATCTTCCT GATGAGCTACGACTTCTATG GCGCCTTCGA TCTGAAGAAC 1560 CTGGGGCATC AGACCGCGCT GAATGCGCCGGCCTGGAAAC CGGACACCGC CTACACCACG 1620 GTGAACGGCG TCAATGCGCT GCTGGCGCAGGGCGTCAAGC CGGGCAAAAT CGTCGTCGGC 1680 ACCGCCATGT ATGGCCGCGG CTGGACCGGGGTGAACGGCT ACCAGAACAA TATTCCGTTC 1740 ACCGGCACCG CCACCGGGCC GGTTAAAGGCACCTGGGAGA ACGGTATCGT GGACTACCGC 1800 CAAATCGCCG GCCAGTTCAT GAGCGGCGAGTGGCAGTATA CCTACGACGC CACGGCGGAA 1860 GCGCCTTACG TGTTCAAACC TTCCACCGGCGATCTGATCA CCTTCGACGA TGCCCGCTCG 1920 GTGCAGGCTA AAGGCAAGTA CGTGTTGGATAAGCAGCTGG GCGGCCTGTT CTCCTGGGAG 1980 ATCGACGCGG ATAACGGCGA TATTCTCAACAGCATGAACG CCAGCCTGGG CAACAGCGCC 2040 GGCGTTCAAT AATCGGTTGC AGTGGTTGCCGGGGGATATC CTTTCGCCCC CGGCTTTTTC 2100 GCCGACGAAA GTTTTTTTAC GCCGCACAGATTGTGGCTCT GCCCCGAGCA AAACGCGCTC 2160 ATCGGACTCA CCCTTTTGGG TAATCCTTCAGCATTTCCTC CTGTCTTTAA CGGCGATCAC 2220 AAAAATAACC GTTCAGATAT TCATCATTCAGCAACAAAGT TTTGGCGTTT TTTAACGGAG 2280 TTAAAAACCA GTAAGTTTGT GAGGGTCAGACCAATGCGCT AAAAATGGG 2329 1002 base pairs nucleic acid single linearcDNA Hordeum vulgare L. 5′UTR 1..63 CDS 64..861 /codon_start= 64/function=“chitinase” /product=“26 kD preprotein of chitinase G (ChiG)”/note= “antifungal activity, especially on Trichoderma reesii andFusarium sporotrichoides as well as Rhizoctonia solani and Botrytiscinerea.” 3′UTR 862..1002 /partial /note= “11 nucleotides at 3′ end notshown” polyA_signal 905..910 /note= “potential polyadenylation signal”sig_peptide 64..294 /note= “probable signal peptide sequence”sig_peptide 298..312 /note= “probable signal peptide sequence”sig_peptide 349..378 /note= “probable signal peptide sequence”sig_peptide 466..588 /note= “probable signal peptide sequence”sig_peptide 607..861 /note= “probable signal peptide sequence”mat_peptide 133..861 9 CCTACGACAG TAGCGTAACG GTAAACACCG AGTACGGTACTCTGTGCTTT GTTGGCTCGC 60 ACA ATG AGA TCG CTC GCG GTG GTG GTG GCC GTG GTAGCC ACG GTG GCC 108 Met Arg Ser Leu Ala Val Val Val Ala Val Val Ala ThrVal Ala -23 -20 -15 -10 ATG GCC ATC GGC ACG GCG CGC GGC AGC GTG TCC TCCATC GTC TCG CGC 156 Met Ala Ile Gly Thr Ala Arg Gly Ser Val Ser Ser IleVal Ser Arg -5 1 5 GCA CAG TTT GAC CGC ATG CTT CTC CAC CGC AAC GAC GGCGCC TGC CAG 204 Ala Gln Phe Asp Arg Met Leu Leu His Arg Asn Asp Gly AlaCys Gln 10 15 20 GCC AAG GGC TTC TAC ACC TAC GAC GCC TTC GTC GCC GCC GCAGCC GCC 252 Ala Lys Gly Phe Tyr Thr Tyr Asp Ala Phe Val Ala Ala Ala AlaAla 25 30 35 40 TTC CCG GGC TTC GGC ACC ACC GGC AGC GCC GAC GCC CAG AAGCGC GAG 300 Phe Pro Gly Phe Gly Thr Thr Gly Ser Ala Asp Ala Gln Lys ArgGlu 45 50 55 GTG GCC GCC TTC CTA GCA CAG ACC TCC CAC GAG ACC ACC GGC GGGTGG 348 Val Ala Ala Phe Leu Ala Gln Thr Ser His Glu Thr Thr Gly Gly Trp60 65 70 GCG ACT GCA CCG GAC GGG GCC TTC GCC TGG GGC TAC TGC TTC AAG CAG396 Ala Thr Ala Pro Asp Gly Ala Phe Ala Trp Gly Tyr Cys Phe Lys Gln 7580 85 GAA CGT GGC GCC TCC TCC GAC TAC TGC ACC CCG AGC GCA CAA TGG CCG444 Glu Arg Gly Ala Ser Ser Asp Tyr Cys Thr Pro Ser Ala Gln Trp Pro 9095 100 TGC GCC CCC GGG AAG CGC TAC TAC GGC CGC GGG CCA ATC CAG CTC TCC492 Cys Ala Pro Gly Lys Arg Tyr Tyr Gly Arg Gly Pro Ile Gln Leu Ser 105110 115 120 CAC AAC TAC AAC TAT GGA CCT GCC GGC CGG GCC ATC GGG GTC GATCTG 540 His Asn Tyr Asn Tyr Gly Pro Ala Gly Arg Ala Ile Gly Val Asp Leu125 130 135 CTG GCC AAC CCG GAC CTG GTG GCC ACG GAC GCC ACT GTG GGC TTTAAG 588 Leu Ala Asn Pro Asp Leu Val Ala Thr Asp Ala Thr Val Gly Phe Lys140 145 150 ACG GCC ATC TGG TTC TGG ATG ACG GCG CAG CCG CCC AAG CCA TCGAGC 636 Thr Ala Ile Trp Phe Trp Met Thr Ala Gln Pro Pro Lys Pro Ser Ser155 160 165 CAT GCT GTG ATC GCC GGC CAG TGG AGC CCG TCA GGG GCT GAC CGGGCC 684 His Ala Val Ile Ala Gly Gln Trp Ser Pro Ser Gly Ala Asp Arg Ala170 175 180 GCA GGC CGG GTG CCC GGG TTT GGT GTG ATC ACC AAC ATC ATC AACGGC 732 Ala Gly Arg Val Pro Gly Phe Gly Val Ile Thr Asn Ile Ile Asn Gly185 190 195 200 GGG ATC GAG TGC GGT CAC GGG CAG GAC AGC CGC GTC GCC GATCGA ATC 780 Gly Ile Glu Cys Gly His Gly Gln Asp Ser Arg Val Ala Asp ArgIle 205 210 215 GGG TTT TAC AAG CGC TAC TGT GAC ATC CTC GGC GTT GGC TACGGC AAC 828 Gly Phe Tyr Lys Arg Tyr Cys Asp Ile Leu Gly Val Gly Tyr GlyAsn 220 225 230 AAC CTC GAT TGC TAC AGC CAG AGA CCC TTC GCC TAATTAATTAGTCATGTATT 881 Asn Leu Asp Cys Tyr Ser Gln Arg Pro Phe Ala 235 240AATCTTGGCC CTCCATAAAA TACAATAAGA GCATCGTCTC CTATCTACAT GCTGTAAGAT 941GTAACTATGG TAACCTTTTA TGGGGAACAT AACAAAGGCA TCTCGTATAG ATGCTTTGCT 1001 A1002 266 amino acids amino acid linear protein not provided 10 Met ArgSer Leu Ala Val Val Val Ala Val Val Ala Thr Val Ala Met -23 -20 -15 -10Ala Ile Gly Thr Ala Arg Gly Ser Val Ser Ser Ile Val Ser Arg Ala -5 1 5Gln Phe Asp Arg Met Leu Leu His Arg Asn Asp Gly Ala Cys Gln Ala 10 15 2025 Lys Gly Phe Tyr Thr Tyr Asp Ala Phe Val Ala Ala Ala Ala Ala Phe 30 3540 Pro Gly Phe Gly Thr Thr Gly Ser Ala Asp Ala Gln Lys Arg Glu Val 45 5055 Ala Ala Phe Leu Ala Gln Thr Ser His Glu Thr Thr Gly Gly Trp Ala 60 6570 Thr Ala Pro Asp Gly Ala Phe Ala Trp Gly Tyr Cys Phe Lys Gln Glu 75 8085 Arg Gly Ala Ser Ser Asp Tyr Cys Thr Pro Ser Ala Gln Trp Pro Cys 90 95100 105 Ala Pro Gly Lys Arg Tyr Tyr Gly Arg Gly Pro Ile Gln Leu Ser His110 115 120 Asn Tyr Asn Tyr Gly Pro Ala Gly Arg Ala Ile Gly Val Asp LeuLeu 125 130 135 Ala Asn Pro Asp Leu Val Ala Thr Asp Ala Thr Val Gly PheLys Thr 140 145 150 Ala Ile Trp Phe Trp Met Thr Ala Gln Pro Pro Lys ProSer Ser His 155 160 165 Ala Val Ile Ala Gly Gln Trp Ser Pro Ser Gly AlaAsp Arg Ala Ala 170 175 180 185 Gly Arg Val Pro Gly Phe Gly Val Ile ThrAsn Ile Ile Asn Gly Gly 190 195 200 Ile Glu Cys Gly His Gly Gln Asp SerArg Val Ala Asp Arg Ile Gly 205 210 215 Phe Tyr Lys Arg Tyr Cys Asp IleLeu Gly Val Gly Tyr Gly Asn Asn 220 225 230 Leu Asp Cys Tyr Ser Gln ArgPro Phe Ala 235 240 1235 base pairs nucleic acid single linear cDNAHordeum vulgare L. 5′UTR 1..48 CDS 49..1050 /partial /codon_start= 49/function=“glucanase” /product=“preprotein of the glucanase GluG” 3′UTR1051..1235 /partial /note= “14 nucleotides at the 3′end not shown.”polyA_signal 1083..1088 /note= “potential polyadenylation signal”polyA_signal 1210..1215 /note= “potential polyadenylation signal”mat_peptide 133..1050 11 GGCAGCATTG CATAGCATTT GAGCACCAGA TACTCCGTGTGTGCACCA ATG GCT AGA 57 Met Ala Arg -28 AAA GAT GTT GCC TCC ATG TTT GCAGTT GCT CTC TTC ATT GGA GCA TTC 105 Lys Asp Val Ala Ser Met Phe Ala ValAla Leu Phe Ile Gly Ala Phe -25 -20 -15 -10 GCT GCT GTT CCT ACG AGT GTGCAG TCC ATC GGC GTA TGC TAC GGC GTG 153 Ala Ala Val Pro Thr Ser Val GlnSer Ile Gly Val Cys Tyr Gly Val -5 1 5 ATC GGC AAC AAC CTC CCC TCC CGGAGC GAC GTG GTG CAG CTC TAC AGG 201 Ile Gly Asn Asn Leu Pro Ser Arg SerAsp Val Val Gln Leu Tyr Arg 10 15 20 TCC AAG GGC ATC AAC GGC ATG CGC ATCTAC TTC GCC GAC GGG CAG GCC 249 Ser Lys Gly Ile Asn Gly Met Arg Ile TyrPhe Ala Asp Gly Gln Ala 25 30 35 CTC TCG GCC GTC CGC AAC TCC GGC ATC GGCCTC ATC CTC GAC ATC GGC 297 Leu Ser Ala Val Arg Asn Ser Gly Ile Gly LeuIle Leu Asp Ile Gly 40 45 50 55 AAC GAC CAG CTC GCC AAC ATC GCC GCC AGCACC TCC AAC GCG GCC TCC 345 Asn Asp Gln Leu Ala Asn Ile Ala Ala Ser ThrSer Asn Ala Ala Ser 60 65 70 TGG GTC CAG AAC AAC GTG CGG CCC TAC TAC CCTGCC GTG AAC ATC AAG 393 Trp Val Gln Asn Asn Val Arg Pro Tyr Tyr Pro AlaVal Asn Ile Lys 75 80 85 TAC ATC GCC GCC GGC AAC GAG GTG CAG GGC GGC GCCACG CAG AGC ATC 441 Tyr Ile Ala Ala Gly Asn Glu Val Gln Gly Gly Ala ThrGln Ser Ile 90 95 100 CTG CCG GCC ATG CGC AAC CTC AAC GCG GCC CTC TCCGCG GCG GGG CTC 489 Leu Pro Ala Met Arg Asn Leu Asn Ala Ala Leu Ser AlaAla Gly Leu 105 110 115 GGC GCC ATC AAG GTG TCC ACC TCC ATC CGG TTC GACGAG GTG GCC AAC 537 Gly Ala Ile Lys Val Ser Thr Ser Ile Arg Phe Asp GluVal Ala Asn 120 125 130 135 TCC TTC CCG CCC TCC GCC GGC GTG TTC AAG AACGCC TAC ATG ACG GAC 585 Ser Phe Pro Pro Ser Ala Gly Val Phe Lys Asn AlaTyr Met Thr Asp 140 145 150 GTG GCC CGG CTC CTG GCG AGC ACC GGC GCG CCGCTG CTC GCC AAC GTC 633 Val Ala Arg Leu Leu Ala Ser Thr Gly Ala Pro LeuLeu Ala Asn Val 155 160 165 TAC CCC TAC TTC GCG TAC CGT GAC AAC CCC GGGAGC ATC AGC CTG AAC 681 Tyr Pro Tyr Phe Ala Tyr Arg Asp Asn Pro Gly SerIle Ser Leu Asn 170 175 180 TAC GCG ACG TTC CAG CCG GGC ACC ACC GTG CGTGAC CAG AAC AAC GGG 729 Tyr Ala Thr Phe Gln Pro Gly Thr Thr Val Arg AspGln Asn Asn Gly 185 190 195 CTG ACC TAC ACG TCC CTG TTC GAC GCG ATG GTGGAC GCC GTG TAC GCG 777 Leu Thr Tyr Thr Ser Leu Phe Asp Ala Met Val AspAla Val Tyr Ala 200 205 210 215 GCG CTG GAG AAG GCC GGC GCG CCG GCG GTGAAG GTG GTG GTG TCG GAG 825 Ala Leu Glu Lys Ala Gly Ala Pro Ala Val LysVal Val Val Ser Glu 220 225 230 AGC GGG TGG CCG TCG GCG GGC GGG TTT GCGGCG TCG GCC GGC AAT GCG 873 Ser Gly Trp Pro Ser Ala Gly Gly Phe Ala AlaSer Ala Gly Asn Ala 235 240 245 CGG ACG TAC AAC CAG GGG CTG ATC AAC CACGTC GGC GGG GGC ACG CCC 921 Arg Thr Tyr Asn Gln Gly Leu Ile Asn His ValGly Gly Gly Thr Pro 250 255 260 AAG AAG CGG GAG GCG CTG GAG ACG TAC ATCTTC GCC ATG TTC AAC GAG 969 Lys Lys Arg Glu Ala Leu Glu Thr Tyr Ile PheAla Met Phe Asn Glu 265 270 275 AAC CAG AAG ACC GGG GAC GCC ACG GAG AGGAGC TTC GGG CTC TTC AAC 1017 Asn Gln Lys Thr Gly Asp Ala Thr Glu Arg SerPhe Gly Leu Phe Asn 280 285 290 295 CCG GAC AAG TCG CCG GCA TAC AAC ATCCAG TTC TAGTACGTGT AGCTACCTAG 1070 Pro Asp Lys Ser Pro Ala Tyr Asn IleGln Phe 300 305 CTCACATACC TAAATAAATA AGCTGCACGT ACGTACGTAA TGCGGCATCCAAGTGTAACG 1130 TAGACACGTA CATTCATCCA TGGAAGAGTG CAACCAAGCA TGCGTTAACTTCCTGGTGAT 1190 GATACATCAT CATGGTATGA ATAAAAGATA TGGAAGATGT TATGA 1235334 amino acids amino acid linear protein not provided 12 Met Ala ArgLys Asp Val Ala Ser Met Phe Ala Val Ala Leu Phe Ile -28 -25 -20 -15 GlyAla Phe Ala Ala Val Pro Thr Ser Val Gln Ser Ile Gly Val Cys -10 -5 1 TyrGly Val Ile Gly Asn Asn Leu Pro Ser Arg Ser Asp Val Val Gln 5 10 15 20Leu Tyr Arg Ser Lys Gly Ile Asn Gly Met Arg Ile Tyr Phe Ala Asp 25 30 35Gly Gln Ala Leu Ser Ala Val Arg Asn Ser Gly Ile Gly Leu Ile Leu 40 45 50Asp Ile Gly Asn Asp Gln Leu Ala Asn Ile Ala Ala Ser Thr Ser Asn 55 60 65Ala Ala Ser Trp Val Gln Asn Asn Val Arg Pro Tyr Tyr Pro Ala Val 70 75 80Asn Ile Lys Tyr Ile Ala Ala Gly Asn Glu Val Gln Gly Gly Ala Thr 85 90 95100 Gln Ser Ile Leu Pro Ala Met Arg Asn Leu Asn Ala Ala Leu Ser Ala 105110 115 Ala Gly Leu Gly Ala Ile Lys Val Ser Thr Ser Ile Arg Phe Asp Glu120 125 130 Val Ala Asn Ser Phe Pro Pro Ser Ala Gly Val Phe Lys Asn AlaTyr 135 140 145 Met Thr Asp Val Ala Arg Leu Leu Ala Ser Thr Gly Ala ProLeu Leu 150 155 160 Ala Asn Val Tyr Pro Tyr Phe Ala Tyr Arg Asp Asn ProGly Ser Ile 165 170 175 180 Ser Leu Asn Tyr Ala Thr Phe Gln Pro Gly ThrThr Val Arg Asp Gln 185 190 195 Asn Asn Gly Leu Thr Tyr Thr Ser Leu PheAsp Ala Met Val Asp Ala 200 205 210 Val Tyr Ala Ala Leu Glu Lys Ala GlyAla Pro Ala Val Lys Val Val 215 220 225 Val Ser Glu Ser Gly Trp Pro SerAla Gly Gly Phe Ala Ala Ser Ala 230 235 240 Gly Asn Ala Arg Thr Tyr AsnGln Gly Leu Ile Asn His Val Gly Gly 245 250 255 260 Gly Thr Pro Lys LysArg Glu Ala Leu Glu Thr Tyr Ile Phe Ala Met 265 270 275 Phe Asn Glu AsnGln Lys Thr Gly Asp Ala Thr Glu Arg Ser Phe Gly 280 285 290 Leu Phe AsnPro Asp Lys Ser Pro Ala Tyr Asn Ile Gln Phe 295 300 305

1. Transgenic pathogen-resistant organism characterized in that itsgenome contains at least two different genes under the control of activepromoters with pathogen-inhibiting action.
 2. Transgenicpathogen-resistant organism according to claim 1 , characterized in thatthe genes code for gene products which reduce the vitality of fungi. 3.Transgenic pathogen-resistant organism according to claim 1 or 2 ,characterized in that the genes are of fungal, bacterial, plant, animalor viral origin.
 4. Transgenic pathogen-resistant organism according toclaim 2 or 3 , characterized in that the gene products have propertiespromoting resistance to fungi.
 5. Transgenic pathogen-resistant organismaccording to claim 4 , characterized in that the gene products arechitinase (ChiS, ChiG), glucanase (GluG), protein synthesis inhibitor(PSI) and antifungal protein (AFP).
 6. Transgenic pathogen-resistantorganism according to any of claims 1 to 5 , characterized in that thelatter is a plant.
 7. Transgenic pathogen-resistant organism accordingto claim 6 , characterized in that it is a tobacco, potato, strawberry,corn, rape or tomato plant.
 8. DNA-transfer vectors with inserted DNAsequences according to one or more of the preceding claims.
 9. Processfor the generation of pathogen-resistant organisms according to any ofclaims 1-7, characterized in that at least one gene withpathogen-inhibiting action is transferred into the genome of anorganism, and the pathogen-resistant organism is obtained (a) bycrossing the organism with another, optionally transgenic, organismwhich contains at least one other gene with pathogen-inhibiting action,and subsequently selecting, and/or (b) by transformation of at least oneother gene with pathogen-inhibiting action into the organism. 10.Process according to claim 9 , characterized in that DNA-transfervectors with inserted DNA sequences corresponding to a gene withpathogen-inhibiting action as described in any of claims 1 to 5 areused.
 11. Process for the generation of pathogen-resistant organismsaccording to any of claims 1-7, characterized in that vectors whichcomprises more than one gene with pathogen-inhibiting action are usedfor the transformation into the genome of an organism.
 12. Process forensuring the resistance of organisms to pathogens, characterized in thatthe organism used is a transgenic pathogen-resistant organism accordingto any of claims 1 to 7 or an organism whose genome contains at leastone gene complying with the definitions of claims 1 to 7 , and at leastone substance which is not expressed by the organism but corresponds toany other one of the gene products complying with claims 1 to 7 isapplied to the organism.